WO2015037535A1 - 人工多能性幹細胞の分化誘導方法及び選別方法 - Google Patents
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Definitions
- the present invention relates to a method for inducing differentiation of induced pluripotent stem cells, a method for selecting induced pluripotent stem cells, and a kit for selecting induced pluripotent stem cells.
- Stem cells play an important role in regenerative medicine.
- Stem cells with universal differentiation include embryonic stem cells (ES cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), nuclear transplant ES cells, somatic cell-derived ES cells (ntES cells) and Artificial pluripotent stem cells (iPS cells) are known, and somatic stem cells, tissue stem cells, and adult stem cells are known as stem cells having pluripotent pluripotency.
- iPS cells induced pluripotent stem cells
- iPS cells are pluripotent and have no ethical problems associated with the destruction of embryos and eggs because they are artificially produced from somatic cells. Since there is no problem of compatibility at the time of transplantation, application to regenerative medicine is expected.
- iPS cells induced pluripotent stem cells
- pancreatic cells pancreatic cells, hepatocytes, cardiomyocytes, blood cells, germ cells, nerve cells, etc.
- iPS cells induced pluripotent stem cells
- Patent Document 1 discloses a method for inducing differentiation of a stem cell into a specific cell line, in which the stem cell is differentiated into a specific cell line in the presence of a tissue sample and / or an extracellular medium of the tissue sample. A method is described that includes culturing in vitro under inducing conditions, wherein the differentiated stem cells are of the same cell type as the tissue sample.
- the stem cells used in Patent Document 1 are embryonic stem cells (ES cells), and there is no description regarding induced pluripotent stem cells (iPS cells).
- Another problem is to establish a method for evaluating and managing the quality of iPS cells. That is, the present invention provides a method for efficiently inducing differentiation of iPS cells into cells having a target function, and a method for selecting iPS cells having high differentiation induction efficiency into target cells from the prepared iPS cells. It was set as a problem to be solved. Furthermore, another object of the present invention is to provide an iPS cell sorting kit for use in the above method.
- the present inventors have conducted intensive research to solve the above-mentioned problems, and found that differentiation induction into a target cell can be achieved by culturing iPS cells on a frozen section of a tissue / organ targeted for regeneration.
- the invention has been completed.
- a method for inducing differentiation of an induced pluripotent stem cell comprising culturing the induced pluripotent stem cell on a structure containing cells and / or a component derived from the cell, and induction of differentiation with the cells in the structure
- the differentiation induction method as described above, wherein the differentiated cell is the same cell type.
- the method according to (1), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
- the method according to (1) or (2), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
- a method for producing a differentiation-induced cell comprising culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, and induction of differentiation with the cell in the structure
- the above production method wherein the prepared cells are of the same cell type.
- the structure containing cells and / or components derived from cells is a sheet-like structure.
- the method according to (6) or (7), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
- the method according to any one of (5) to (7), wherein the cell is liver, brain or spinal cord.
- a method for selecting induced pluripotent stem cells comprising the steps of: a cell in a structure and a cell induced to differentiate are of the same cell type.
- the method according to (9), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
- the method according to (9) or (10), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ section or a cell.
- the method according to any one of (9) to (11), wherein the cells are liver, brain or spinal cord.
- a kit for selecting induced pluripotent stem cells comprising at least a structure containing cells and / or components derived from cells.
- the cell and / or the structure containing the component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
- the kit according to any one of (13) to (15), wherein the cells are liver, brain or spinal cord.
- the method of the present invention it is possible to induce differentiation of iPS cells into cells having a target function with high efficiency. Furthermore, according to the method of the present invention, it is possible to select iPS cells having high efficiency of inducing differentiation into target cells from the prepared iPS cells.
- FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on cover glass (control), normal liver section, or hepatitis liver section.
- FIG. 2 shows microscopic images (3rd and 9th day of culture) of iPS cells cultured on cover glass (control) or normal liver sections.
- FIG. 3 shows microscopic images of iPS cells cultured on brain sections or spinal cord sections (3rd day and 9th day of culture).
- FIG. 4 shows the results of examining the expression of genes related to hepatocytes (AFP, AAT, ALB) by RT-PCR.
- FIG. 5 shows the results of examining the expression of genes related to neurons (Nestin, MBP, CNPase, and GFAP) by RT-PCR.
- FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on cover glass (control), normal liver section, or hepatitis liver section.
- FIG. 2 shows microscopic images (3
- FIG. 6 shows the results of immunocytochemical analysis of AFP expression.
- FIG. 7 shows the results of measuring the ratio of AFP positive cells in nucleated cells.
- FIG. 8 shows the results of immunocytochemical analysis of AAT expression.
- FIG. 9 shows the results of measuring the ratio of AAT positive cells in nucleated cells.
- FIG. 10 shows the results of immunocytochemical analysis of GFAP expression.
- FIG. 11 shows the results of measuring the ratio of GFAP positive cells in nucleated cells.
- FIG. 12 shows the results of immunocytochemical analysis of CNPase expression.
- FIG. 13 shows the results of immunocytochemical analysis of CNPase expression.
- FIG. 14 shows the results of measuring the ratio of CNPase positive cells in nucleated cells.
- the present invention comprises culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell.
- the cells in the structure and the cells induced to differentiate are of the same cell type. Whether cells in the structure and differentiated cells are the same cell type can be determined by, for example, the same marker to be expressed.
- Cell markers for hepatocytes include ⁇ -fetoprotein (AFP), ⁇ -1 antitrypsin (AAT), albumin (ALB), tyrosine aminotransferase (TAT), tryptophan 2,3 dioxygenase (TDO2), cytochrome P450, etc.
- AFP ⁇ -fetoprotein
- AAT ⁇ -1 antitrypsin
- AAT albumin
- TAT tyrosine aminotransferase
- TDO2 tryptophan 2,3 dioxygenase
- Neuronal markers include nestin Nestin, myelin basic protein (MBP), cyclic nucleotide phosphodiesterase (CNPase), glial fibrillary acidic protein (GFAP), and neurofilament (Neurofilament), but are particularly limited Not.
- MBP myelin basic protein
- CNPase cyclic nucleotide phosphodiesterase
- GFAP glial fibrillary
- osteoblast markers include, but are not limited to, alkaline phosphatase (ALP), osteopontin, and osteocalcin.
- pancreatic cell markers include, but are not limited to, Pdx1, amylase, and carboxypeptidase.
- chondrocyte markers include, but are not limited to, Sox9, type II collagen, and aggrecan.
- cardiomyocyte markers include cardiac troponin I (cTnI), ⁇ -myosin heavy chain ( ⁇ -MHC), ⁇ cardiac actin ( ⁇ -cardiac actin), and homeobox protein Nkx-2.5. However, it is not particularly limited.
- the form of the “structure containing a cell and / or a component derived from the cell” used in the present invention is not particularly limited, in view of culturing induced pluripotent stem cells (iPS cells) on the structure, a sheet It is preferable that it is a structure.
- a culture substrate coated with a component derived from a living tissue, an organ section or a cell can be used as an example of the sheet-like structure containing a cell and / or a component derived from a cell.
- the thickness of the sheet-like structure containing cells is not particularly limited, but is generally about 1 to 100 ⁇ m, preferably about 2 to 50 ⁇ m, more preferably It is about 2 to 20 ⁇ m.
- the form of the “culture substrate” used in the present invention is not particularly limited, but is preferably a film, a plate or a cover glass.
- Tissue or organ sections can be preferably collected from mammals (preferably mice or humans).
- the type of tissue or organ is not particularly limited, and a tissue or cell containing cells of the same cell type as a differentiated cell type that is induced to differentiate from an induced pluripotent stem cell (iPS cell)
- An organ section may be used.
- tissues or organs include, but are not limited to, liver, brain, spinal cord, heart, respiratory organ, reproductive organ, kidney, pancreas, skin, muscle, and skeletal organ.
- a liver slice may be used.
- a slice containing nerve cells may be used.
- components derived from cells include nucleic acids (DNA or RNA, particularly micro RNA) or proteins, and particularly nucleic acids (DNA or RNA, particularly micro RNA) or specifically expressed in the cells or Protein is preferred.
- the induced pluripotent stem cell is transferred to a specific cell lineage, preferably a cell such as liver, nerve, lung, prostate, pancreas, mammary gland, kidney, intestine, skeleton, blood vessel, hematopoiesis, heart muscle, skeletal muscle, etc. Differentiation into lineages is induced.
- a specific cell lineage preferably a cell such as liver, nerve, lung, prostate, pancreas, mammary gland, kidney, intestine, skeleton, blood vessel, hematopoiesis, heart muscle, skeletal muscle, etc. Differentiation into lineages is induced.
- artificial pluripotent stem cells are established by introducing 4 genes of OCT3 / 4, SOX2, NANOG, and LIN28 into human fibroblasts (Yu J., Thomson JA.et al., Science). (2007) 318: 1917-1920.).
- an artificial pluripotent stem cell is established by introducing 6 genes of OCT3 / 4, SOX2, KLF4, C-MYC, hTERT, SV40 large T into skin cells (Park IH, Daley GQ.et al., Nature (2007) 451: 141-146), establishing induced pluripotent stem cells by introducing Oct3 / 4, Sox2, Klf4, c-Myc, etc. into undifferentiated stem cells present in postnatal tissues rather than somatic cells (Japanese Patent Laid-Open No. 2008-307007) has also been reported.
- an induced pluripotent stem cell is a cell having multipotency and self-proliferation ability induced by reprogramming somatic cells or undifferentiated stem cells.
- the origin of the somatic cell is not limited, and may be derived from an embryo, fetus, or adult.
- the animal species from which the somatic cell is derived is not particularly limited, but is preferably a mammal, more preferably a human or a mouse.
- somatic cells include, but are not limited to, fibroblasts, epithelial cells, hepatocytes, blood cells and the like.
- the method for producing induced pluripotent stem cells used in the present invention is not particularly limited, and the introduction factor and the introduction method are not particularly limited.
- the feeder cells are detached and iPS cells are collected, and an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM) containing fetal calf serum) Suspend in Next, a culture cover glass on which a structure containing cells (preferably, a section of a living tissue or an organ) is placed may be placed, and an iPS cell suspension may be seeded therein.
- DMEM Dulbecco's modified Eagle medium
- FBS fetal bovine serum
- the conditions for culturing induced pluripotent stem cells on a structure containing cells and / or components derived from cells are not particularly limited as long as differentiation of induced pluripotent stem cells can be induced.
- the medium for example, Dulbecco's modified Eagle medium (DMEM) containing fetal bovine serum, or a medium for primate ES cells (Reprocell Co., Ltd.) can be used.
- DMEM Dulbecco's modified Eagle medium
- Various growth factors for inducing differentiation of induced pluripotent stem cells, cytokines or differentiation inducing factors may be added and cultured.
- by culturing on a structure containing cells. Differentiation induction can be achieved without adding the above-mentioned growth factors and differentiation induction factors.
- Examples of various growth factors, cytokines or differentiation inducing factors for inducing differentiation of induced pluripotent stem cells include actin bin, bFGF, noggin, nicotinamide, retinoic acid, EGF, glucocorticoid, etc. There is no particular limitation.
- induced pluripotent stem cells are induced to differentiate by culturing induced pluripotent stem cells on a structure containing cells and / or components derived from the cells.
- induced pluripotent stem cells with high differentiation induction efficiency can be selected.
- induced pluripotent stem cells are currently produced by various methods.
- the created induced pluripotent stem cells are expected to be induced to differentiate into desired cells and applied to regenerative medicine.
- the created artificial pluripotent stem cells usually contain a mixture of cells having high efficiency of inducing differentiation into desired cells and cells having low efficiency of inducing differentiation into desired cells. . It has been desired to select cells with high efficiency of inducing differentiation into desired cells from the prepared induced pluripotent stem cells.
- an induced pluripotent stem cell is induced to differentiate by culturing the induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, and the efficiency of inducing differentiation into a desired cell is improved.
- Highly induced pluripotent stem cells can be selected.
- the induced pluripotent stem cells selected in this way and having high differentiation-inducing efficiency can be stored as a stock, and can be differentiated and used when necessary in the field of regenerative medicine.
- a structure containing cells and / or components derived from cells such as a section of a living tissue or an organ, can be provided as a kit for selecting induced pluripotent stem cells.
- the kit suitably includes a medium for culturing induced pluripotent stem cells, a cover glass for installing structures containing cells, and the like. May be.
- iPS cell culture and differentiation induction After culturing iPS cells according to a conventional method, the feeder cells are detached using an iPS / ES cell detachment solution (CTK solution, Reprocell Co., Ltd.), washed with PBS, and then iPS cells. Were collected and suspended in Dulbecco's modified Eagle medium (hereinafter abbreviated as DMEM, SIGMA) containing 5% fetal bovine serum (hereinafter abbreviated as FBS, Biological Industries). A circular culture cover glass on which the aforementioned frozen section was placed was placed on a 35 mm diameter dish (non-coated type, Matsunami glass), and a suspension of iPS cells was seeded thereon.
- CTK solution iPS / ES cell detachment solution
- FBS fetal bovine serum
- FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on a cover glass (control), a normal liver section, or a hepatitis liver section. It was observed that the colonies of iPS cells seeded in the control, normal liver, and hepatitis liver spread and the cells spread. In addition, changes in cell morphology were also observed. In the control, iPS cells that had expanded on the 8th day compared to the 2nd day of culture exhibited various forms as indicated by arrows, and no uniformity was observed. On the other hand, in the normal liver and hepatitis liver, the expanded cells showed a relatively large and polygonal shape as indicated by arrows.
- FIG. 2 a microscopic image (3rd and 9th culture days) of iPS cells cultured on a cover glass (control) or a normal liver slice is shown in FIG. 2, and the iPS cells cultured on a brain slice or spinal cord slice are shown.
- a microscopic image (3rd day and 9th day of culture) is shown in FIG.
- RNA expression of various differentiation markers of neurons and hepatocytes was collected from iPS cells cultured on frozen sections of liver, brain, and spinal cord using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA).
- RNA obtained 1 ⁇ g was treated with 100 units / ml deoxyribonuclease I (hereinafter abbreviated as DNase I) at room temperature, and then 1 ml of 25 mM EDTA was added and treated at 65 ° C. for 5 minutes to lose DNase I. I made it live.
- a random hexamer primer (Invitrogen) was added, and a reverse transcription reaction was performed at 50 ° C. for 50 minutes using the reverse transcriptase SUPERSCRIPT III Preamplification System (Invitrogen). Further, it was treated with ribonuclease H at 37 ° C. for 20 minutes to obtain cDNA.
- PCR was performed using primers specific to each human gene and PCR MasterMix Kit (Thermo, Rockford, USA) to examine mRNA expression of each gene.
- the examined differentiation markers, primers and PCR conditions are shown in Table 1.
- PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and then visualized using a UV imaging device FAS-III (Toyobo, Osaka).
- hepatocyte-related genes AFP, AAT, ALB
- the expression of AFP was observed in the control group, normal liver group, and hepatitis liver group, but the expression was stronger in the hepatitis liver group than in the control group, and the expression was further enhanced in the normal liver group.
- AAT expression was also observed in the control group, normal liver group, and hepatitis liver group, but the expression was strongly observed in the normal liver group as compared with the control group.
- the expression of ALB was not observed in the control group, whereas the expression was confirmed in the normal liver group and hepatitis liver group, and the expression was stronger in the normal liver group than in the hepatitis liver group. It was.
- GAPDH Glyceraldehyde-3-phosphate dehydrogenase AFP: ⁇ -fetoprotein AAT: ⁇ 1-antitrypsin ALB: Albumin MBP: Myelin basic protein CNPase: 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase GFAP: Glial fibrous acidic protein NES: Nestin
- FITC-conjugated anti-rabbit IgG or FITC-conjugated anti-goat IgG was reacted as a secondary antibody for 1 hour at room temperature.
- nuclei were stained with DAPI and observed with a fluorescence microscope (FIGS. 6 and 8).
- FIG. 6 almost no AFP positive cells were observed in the control group, but many AFP positive cells were observed in the normal liver group and the hepatitis liver group.
- FIG. 8 as in the case of AFP, more AAT positive cells were observed in the normal liver group and the hepatitis liver group than in the control group.
- the differentiation induction efficiency was examined by measuring the number of iPS cells expressing ⁇ -fetoprotein and ⁇ 1-antitrypsin and calculating the proportion of nucleated cells (FIGS. 7 and 9). As shown in FIG. 7, the ratio of AFP positive cells was significantly higher in the normal liver group and the group on the first day of hepatitis liver than in the control group. In addition, as shown in FIG. 9, the proportion of AAT positive cells was significantly higher in the normal liver group and the hepatitis liver 1, 2 and 5 day groups than in the control group.
- IPS cells cultured on various frozen sections were fixed with 2% paraformaldehyde for 1 hour, washed twice with PBS, and then diluted with 0.1% BSA-containing PBS to 1% TRITON X-100 (ICN Biomedical). For 15 minutes. After washing twice with PBS, blocking was performed with 10% normal goat serum for 1 hour. After washing twice with PBS, the mixture was reacted overnight at 4 ° C. with a primary antibody diluted with PBS containing 1% BSA.
- Rabbit anti-Glial Bibrillary Acidic Protein antibody SIGMA, 80-fold dilution
- Rabbit anti-CNPase antibody Abcam, 100-fold dilution
- a secondary antibody Goat anti-rabbit IgG FITC (Wako, diluted 40-fold) was used for GFAP
- Goat anti-rabbit IgG Alexa Invitergen, diluted 100-fold was used for CNPase.
- FIG. 10 shows the results of observation of protein expression with a fluorescence microscope using an anti-human GFAP antibody as a primary antibody.
- the number of iPS cells expressing GFAP was counted, and the proportion of nucleated cells was calculated (FIG. 11), and the differentiation induction efficiency was examined.
- the results of observation of protein expression with a fluorescence microscope using an anti-CNPase antibody as the primary antibody are shown in FIGS.
- the differentiation induction efficiency was examined by measuring the number of iPS cells expressing CNPase and calculating the proportion of nucleated cells (FIG. 14).
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Abstract
Description
(1) 細胞及び/又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む人工多能性幹細胞の分化誘導方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の分化誘導方法。
(2) 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、(1)に記載の方法。
(3) 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、(1)又は(2)に記載の方法。
(4) 細胞が、肝臓、脳又は脊髄である、(1)から(3)の何れかに記載の方法。
(6) 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、(5)に記載の方法。
(7) 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、(6)又は(7)に記載の方法。
(8) 細胞が、肝臓、脳又は脊髄である、(5)から(7)の何れかに記載の方法。
(10) 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、(9)に記載の方法。
(11) 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、(9)又は(10)に記載の方法。
(12) 細胞が、肝臓、脳又は脊髄である、(9)から(11)の何れかに記載の方法。
(14) 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、(13)に記載のキット。
(15) 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、(13)又は(14)に記載のキット。
(16) 細胞が、肝臓、脳又は脊髄である、(13)から(15)の何れかに記載のキット。
(1)人工多能性幹細胞の分化誘導方、並びに分化誘導された細胞の製造方法
本発明は、細胞及び/又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む人工多能性幹細胞の分化誘導方法又は分化誘導された細胞の製造方法に関し、特に、構造物中の細胞と分化誘導された細胞とが同じ細胞型である。なお、構造物中の細胞と分化誘導された細胞とが同じ細胞型であるかどうかは、例えば、発現するマーカーが同じであることにより判断することができる。
神経細胞のマーカーとしては、ネスチンNestin)、ミエリン塩基性蛋白(MBP)、環状ヌクレオチドホスホジエステラーゼ(CNPase)、グリア細胞線維性酸性タンパク質(GFAP)、及びニューロフィラメント(Neurofilament)などが挙げられるが、特に限定されない。
膵臓細胞のマーカーとしては、Pdx1、アミラーゼ、及びカルボキシペプチターゼなどが挙げられるが、特に限定されない。
心筋細胞のマーカーとしては、心筋型トロポニンI(cardiac troponin I:cTnI)、α-ミオシン重鎖(α-MHC)、α心筋アクチン(α-cardiac actin)、及びホメオボックスプロテインNkx-2.5などが挙げられるが、特に限定されない。
細胞に由来する成分を含む構造物は、細胞に由来する成分と相互作用を有する核酸、マイクロRNA、抗体または化合物を用いて細胞に由来する成分を精製し、精製した細胞に由来する成分を培養基材上に付着させ、酵素や界面活性剤等を用いてコーティングさせることで得ることができる。また、コーティングした培養基材をさらに乾燥させることで、長期に保存可能な培養基材を提供できる。
本発明で用いる「培養基材」の形態は特に限定されないが、フィルム、プレートまたはカバーガラスであることが好ましい。
本発明によれば、細胞及び/又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することによって人工多能性幹細胞を分化誘導し、分化誘導効率が高い人工多能性幹細胞を選別することができる。上記した通り、人工多能性幹細胞は現在、様々な方法で作成されている。作成された人工多能性幹細胞は、所望の細胞へと分化誘導され、再生医療などに適用されることが期待されている。しかしながら、作成された人工多能性幹細胞の中には、所望の細胞への分化誘導効率が高い細胞と、所望の細胞への分化誘導効率が低い細胞とが混在しているのが普通である。作成した人工多能性幹細胞の中から所望の細胞への分化誘導効率が高い細胞を選別することが望まれていた。
雄性、6週齢のICRマウスから正常な肝臓、脳および脊髄を摘出した。また、1 ml/kgの四塩化炭素をオリーブ油に1:4の割合で混和したものを腹腔内投与することにより人工的に薬剤性肝炎を発症させたICRマウスから、四塩化炭素投与後1、2、3、5日目に肝炎肝臓を摘出した。
それぞれの臓器をOCTコンパウンドであるTissue-TekR(Sakura Finethechnical)に、包埋、マウントした後、液体窒素に浸漬し凍結切片作成用ブロックを作製した。これらのブロックから、それぞれ厚さ6 μmの凍結切片を作成し、円形のカルチャーカバーグラス(ポリLリジンコートタイプ、松浪ガラス)に載せた。乾燥後、リン酸緩衝液(Phosphate Buffered Saline:以下PBSと略記する、pH 7.4)にて2回洗浄し、OCTコンパウンドを洗い流した後、iPS細胞の培養に用いた。
常法に従いiPS細胞を培養後、iPS/ ES細胞剥離液(CTK solution,株式会社リプロセル)を用いてフィーダー細胞を剥離し、PBSにて洗浄後、iPS細胞を回収し、5%ウシ胎児血清(以下FBSと略記する、Biological Industries)を含むダルベッコ改変イーグル培地(以下DMEMと略記する、SIGMA)に懸濁した。35 mm径のディッシュ(ノンコートタイプ、松浪ガラス)に前述の凍結切片を載せた円形カルチャーカバーグラスを設置し、ここにiPS細胞の懸濁液を播種した。播種後1日間静置することで、浮遊しているiPS細胞を凍結切片へ付着させ、その後5%FBSを含むDMEMを2ml加え培養を行った。その後は3日に1度培地交換を行い、計9日間培養した。
また、カバーグラス上(対照)又は正常肝臓切片上で培養したiPS細胞の顕微鏡像(培養3日目と9日目)を図2に示し、脳切片上又は脊髄切片上で培養したiPS細胞の顕微鏡像(培養3日目と9日目)を図3に示す。
iPS細胞の肝細胞あるいは神経細胞への分化状態を確認するため、神経細胞および肝細胞の各種分化マーカーのmRNA発現について、半定量的Reverse Transcriptase-Polymerase Chain Reaction法(以下RT-PCR法と略する)を用いて、検討を行った。肝臓、脳、脊髄の凍結切片上で培養を行ったiPS細より、TRIzol reagent(Invitrogen Corp.、Carlsbad、 CA、USA)を用いてtotal RNAを回収した。得られたtotal RNA 1μgを室温で100 units/ml デオキシリボヌクレアーゼI(以下DNase Iと略す)で処理した後、1 mlの25 mM EDTAを添加し、65℃、5分間処理し、DNase Iを失活させた。ランダムヘキサマープライマー(Invitrogen)を加え、逆転写酵素SUPERSCRIPT III Preamplification System(Invitrogen)を用いて50℃、50分の条件で逆転写反応を行った。さらに37℃、20分間リボヌクレアーゼHで処理しcDNA を得た。ヒトの各遺伝子に特異的なプライマーおよびPCR MasterMix Kit(Thermo、Rockford、USA)を用いて、PCRを行い各遺伝子のmRNA発現について検討した。検討した分化マーカー、プライマーおよびPCRの条件を表1に示す。PCR産物は2%アガロースゲルを用いて電気泳動を行い、臭化エチジウムで染色後、UV撮影装置FAS-III(東洋紡績、大阪)を用いて可視化した。
AFPの発現は対照群、正常肝臓群、肝炎肝臓群で認められたが、対照群と比較すると肝炎肝臓群で発現が強い傾向がみられ、正常肝臓群ではさらにその発現が増強されていた。AATの発現も対照群、正常肝臓群、肝炎肝臓群で認められたが、対照群と比較すると正常肝臓群で発現が強く認められた。一方、ALBの発現は対照群においては、その発現は認められなかったのに対し、正常肝臓群、肝炎肝臓群では発現が確認され、肝炎肝臓群と比較し正常肝臓群でより強い発現を認めた。この結果から正常肝臓、肝炎肝臓の凍結切片上で培養することによりiPS細胞の固有肝細胞への分化が誘導されていることが確かめられた。
同様に、神経細胞の関連遺伝子(Nestin、MBP、CNPase、及びGFAP)の発現を調べた結果を図5に示す。
AFP:α-フェトプロテイン
AAT:α1-アンチトリプシン
ALB:アルブミン
MBP:ミエリン塩基性蛋白
CNPase:2', 3'-環状ヌクレオチド3'-ホスホジエステラーゼ
GFAP:グリア繊維性酸性タンパク質
NES:ネスチン
各種凍結切片上で培養したiPS細胞をPBSにて洗浄した後、凍結切片ごと、2% パラホルムアルデヒドにて、室温で60分間固定を行った。PBSにて洗浄後、1%Triton-Xにて15分間処理し、10%正常ウサギ血清または正常ヤギ血清にて室温、10分間ブロッキングを行った。その後、ウサギ抗ヒトα-フェトプロテイン抗体(Abcam、500倍希釈)またはヤギ抗ヒトα1-アンチトリプシン抗体(Abcam、300倍希釈)を4℃、一晩反応させた。PBSにて洗浄後、二次抗体としてFITC-conjugated anti-rabbit IgGまたはFITC-conjugated anti-goat IgGを室温で1時間反応させた。PBSにて洗浄後、核をDAPIにて染色し、蛍光顕微鏡にて観察した(図6及び図8)。図6では、対照群ではAFP陽性細胞はほとんど認められなかったが、正常肝臓群、肝炎肝臓群でAFP陽性細胞が多く認められた。図8では、AFPの場合と同様に対照群と比較し正常肝臓群および肝炎肝臓群でAAT陽性細胞が多く認められた。α-フェトプロテインおよびα1-アンチトリプシンを発現しているiPS細胞の数を計測、有核細胞に占める割合を算出することにより(図7及び図9)、分化誘導効率を検討した。図7に示す通り、対照群に比較して正常肝臓群及び肝炎肝臓1日目の群で、AFP陽性細胞の割合が有意に多く認められた。また、図9に示す通り、対照群に比較して、正常肝臓群、肝炎肝臓1,2,5日目の群でAAT陽性細胞の割合が有意に多く認められた。
一次抗体として、抗CNPase抗体を使用してタンパク質発現を蛍光顕微鏡にて観察した結果を図12及び図13に示す。CNPaseを発現しているiPS細胞の数を計測、有核細胞に占める割合を算出することにより(図14)、分化誘導効率を検討した。
Claims (16)
- 細胞及び/又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む人工多能性幹細胞の分化誘導方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の分化誘導方法。
- 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、請求項1に記載の方法。
- 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項1又は2に記載の方法。
- 細胞が、肝臓、脳又は脊髄である、請求項1から3の何れか1項に記載の方法。
- 細胞及び/又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む、分化誘導された細胞の製造方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の製造方法。
- 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、請求項5に記載の方法。
- 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項6又は7に記載の方法。
- 細胞が、肝臓、脳又は脊髄である、請求項5から7の何れか1項に記載の方法。
- 細胞及び/又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することによって人工多能性幹細胞を分化誘導し、分化誘導効率が高い人工多能性幹細胞を選別することを含む人工多能性幹細胞の選別方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の選別方法。
- 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、請求項9に記載の方法。
- 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項9又は10に記載の方法。
- 細胞が、肝臓、脳又は脊髄である、請求項9から11の何れか1項に記載の方法。
- 細胞及び/又は細胞に由来する成分を含む構造物を少なくとも含む、人工多能性幹細胞の選別のためのキット。
- 細胞及び/又は細胞に由来する成分を含む構造物がシート状構造物である、請求項13に記載のキット。
- 細胞及び/又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項13又は14に記載のキット。
- 細胞が、肝臓、脳又は脊髄である、請求項13から15の何れか1項に記載のキット。
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EP3045531B1 (en) | 2023-08-30 |
JP6611170B2 (ja) | 2019-11-27 |
US20160222347A1 (en) | 2016-08-04 |
EP3045531A1 (en) | 2016-07-20 |
EP3045531A4 (en) | 2017-04-19 |
JPWO2015037535A1 (ja) | 2017-03-02 |
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